Summary: Embryos begin development by forming a simple epithelium. Cells fated to become connective tissue then undergo an epithelial-mesenchymal transition (EMT) to leave that early epithelium and populate the interstitial regions of the embryo. This project seeks to understand how the EMT works, specifically how the cells de-adhere from the epithelium, how they become motile to leave the epithelium, and how they invade through the basement membrane beneath the epithelium. Previous work identified the transcriptional subcircuits of the gene regulatory networks responsible for controlling the EMT. In the model system studied, each of the three EMT components ? de-adhesion, motility, and invasion ? are driven by distinct subcircuits of transcription factors. The goal of the project is to identify and functionally understand the effector molecules that are directly regulated downstream of the transcription factors in the subcircuits. Thus, the three aims of the project are: 1) What are the effector molecules, controlled by the de-adhesion subcircuit, that initiate and conduct the deadhesion from the epithelium, and how do they function? 2) What are the effector molecules, controlled by the motility subcircuit, that initiate the motility necessary to move the nascent mesenchymal cell out of the epithelium and through the basal lamina? And, 3) What are the effector molecules, driven by the invasion subcircuit, that are necessary to provide access through the basement membrane for the nascent mesenchyme cells. RNA-seq databases of those targeted cells at different time points of the EMT were obtained, and also RNA-seq databases of the same cells following the same temporal trajectories, but after knockdown of transcription factors in the subcircuits, were obtained to provide a group of candidate effector molecules for the experiments. Three different assays were developed for direct analysis of control and perturbed (knockdowns) of the candidate molecules in an effort to identify participants in controlling the de-adhesion, motility onset, and invasion. In addition to wanting to understand how these three EMT components function in the model system the findings will be pertinent to understand how EMTs operate with high fidelity in embryos to avoid birth defects such as cleft palate. And, the data will be useful to understand progression of disease states where the invasive property similar to normal embryonic cells, is co-opted by cells as they develop metastatic properties.